WO2016118468A1 - Diagnostic de maladie à médiation leucocytaire et exposition à un gaz halogène - Google Patents

Diagnostic de maladie à médiation leucocytaire et exposition à un gaz halogène Download PDF

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WO2016118468A1
WO2016118468A1 PCT/US2016/013834 US2016013834W WO2016118468A1 WO 2016118468 A1 WO2016118468 A1 WO 2016118468A1 US 2016013834 W US2016013834 W US 2016013834W WO 2016118468 A1 WO2016118468 A1 WO 2016118468A1
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gsh
fald
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glutathione
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David A. FORD
Mark A. DUERR
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Saint Louis University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/42—Poisoning, e.g. from bites or stings

Definitions

  • the disclosure relates to the fields of medicine, pathology, biochemistry and cell biology.
  • the disclosure relates to identification and use of glutathione adducts of 2-halofatty aldehydes (FALD-GSH) for leukocyte disease and halogen gas exposure.
  • FLD-GSH 2-halofatty aldehydes
  • eosinophil-driven diseases For example for eosinophilic esophagitis (EE), the only diagnostic tool is taking a biopsy of the esophagitis, followed by obtaining microscopic evidence of eosinophils in a high powered field.
  • EE eosinophilic esophagitis
  • the advantage of a biomarker in a disease such as EE would be to avoid a surgical procedure that requires removing part of the esophagus for pathology microscopic evaluation, sometimes only to find that no disease is present.
  • Other eosinophil-driven conditions that could benefit from improved diagnostics include other gastro-esophageal disease, parasitic infections, sepsis, atherosclerosis, and asthma.
  • the present disclosure provides methods of diagnosing a subject as having or being at risk of developing a leukocyte-mediated disease (LMD) comprising (a) detecting the level of glutathione adducts of 2-halofatty aldehydes (FALD-GSH) in a sample; (b) comparing the amount of FALD-GSH with a control or standard reflective of diseased and/or healthy levels of FALD-GSH; and (c) diagnosing the subject as having or being at risk of developing LMD if the level of FALD-GSH in the sample is higher than the control or standard.
  • the sample may be blood, plasma, serum, sputum, urine, nasal swab, or ear wax.
  • the subject may be suspected of having LMD, may exhibit one or more symptoms of LMD, or may not exhibit a symptom of LMD.
  • the method may further comprise obtaining the sample from the subject. Detecting may comprises (i) mass spectrometry and/or high performance liquid chromatograph (HPLC), or (ii) binding of an antibody to FALD-GSH, such as where an antibody binding to FALD-GSH is employed in immunoprecipitation, Western blot or ELISA.
  • HPLC and mass spectrometry are used in combination.
  • the method may further comprise performing steps (a) and (b) at a second time point to determine progression of LMD or to determine the efficacy of an intervening treatment.
  • the method may further comprise treating the subject if LMD is diagnosed.
  • the LMD may be selected from the group consisting of asthma, sepsis, atherosclerosis, myocardial infarction, eosinophil and neutrophil mediated disease.
  • the standard or control may comprise detected labeled FALD-GSH.
  • the glutathione adduct may be an adduct of 2-bromofatty aldehyde or 2- chlorofatty aldehyde.
  • the glutathione adduct may be hexadecanal glutathione or octadecanal glutathione.
  • a method of diagnosing a subject as having been exposed to a halogen comprising (a) detecting the level of glutathione adducts of 2- halofatty aldehydes (FALD-GSH) in a sample; (b) comparing the amount of FALD-GSH with a control or standard reflective of levels of FALD-GSH from exposed and/or unexposed individuals; and (c) diagnosing the subject as having been exposed to a halogen if the level of FALD-GSH in the sample is higher than the control or standard.
  • FLD-GSH 2- halofatty aldehydes
  • the sample may be blood, plasma, serum, sputum, urine, nasal swab, or ear wax.
  • the subject may be suspected of having been exposed to a halogen, may exhibit one or more symptoms of being exposed to a halogen, or may not exhibit a symptom of being exposed to a halogen.
  • the method may further comprise obtaining the sample from the subject. Detecting comprises (i) mass spectrometry and/or high performance liquid chromatograph (HPLC), or (ii) binding of an antibody to FALD-GSH, such as where an antibody binding to FALD-GSH is employed in immunoprecipitation, Western blot or ELISA. In a particular embodiment, HPLC and mass spectrometry are used in combination.
  • the method may further comprise performing steps (a) and (b) at a second time point to determine continued exposure to a halogen or to determine the efficacy of an intervening treatment.
  • the method may further comprise treating the subject if halogen exposure is diagnosed.
  • the halogen may be is selected from chlorine, bromine, iodine, and fluorine.
  • the standard or control comprises detected labeled FALD-GSH.
  • the glutathione adduct may be an adduct of 2-bromofatty aldehyde or 2-chlorofatty aldehyde.
  • the glutathione adduct may be hexadecanal glutathione or octadecanal glutathione.
  • the terms "about” and “approximately” indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. In one non-limiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • FIGS. 1A-B TLC separation of reaction products from incubations of 2-ClHDA with GSH.
  • GSH (10 mM) was incubated with 2-ClHDA or HDA (20 mM) in the presence or absence of NEM (15 mM) for 4h.
  • Reaction products were resolved by silica TLC, and were visualized by either ninhydrin staining (FIG. 1A) or DNPH staining (FIG. IB).
  • FIGS. 2A-D ESI-MS of TLC-purified 2-ClHDA adduct with GSH.
  • the purity and stability of the TLC-purified reaction product from 2-ClHDA incubations with GSH (HDA-GSH) were confirmed by TLC and ninhydrin staining (FIG. 2A).
  • Lanes 1 and 2 show TLC analysis of the reaction products before and after TLC-purification, respectively.
  • Purified reaction product was analyzed by positive ion (FIG. 2B) and negative ion (FIG. 2C) ESI-MS by direct infusion.
  • FIGS. 3A-B Putative molecular structure of the reaction product from 2-ClHDA and GSH reactions
  • FIGS. 4A-C ESI-MS and MS/MS analysis of DNPH-HDA-GSH HDA-GSH was derivatized with DNPH and analyzed by direct infusion in positive ion mode.
  • FIG. 5 ESI-MS and MS/MS analysis of [ ⁇ /,-] -HDA-GSH.
  • Synthetic [c3 ⁇ 4]-HDA-GSH was analyzed by direct infusion in the positive ion mode with MS/MS analyses as described in FIGS. 3A-B.
  • MS/MS analysis of the [M+H] + ion at m/z 550.35 displays a distinct 4 amu shift compared to the HDA-GSH fragment ions.
  • Survey mode positive ions of synthetic -HDA-GSH are shown in the spectra of the inset.
  • FIGS. 6A-B Stable isotope dilution quantitation of HDA-GSH by LC-ESI- MS/MS. Based on the fragmentation ions shown in FIGS. 3A-B and 4A-C, LC-MS with SRM detection was used to quantify HDA-GSH. Using LC-MS, 15 fmol HDA- GSH and 15 fmol [d 4 ] -HDA-GSH were detected in the SRM scan mode, SRM 546 399 and m/z 550 403, respectively (as indicated in FIG. 6A).
  • FIG. 7 Quantitation of HDA-GSH production in RAW 264.7 cells treated with 2- C1HDA.
  • RAW 264.7 cells were treated with indicated concentrations of 2-ClHDA for 8 hr.
  • FIGS. 8A-B Time course of a-ClFALD and FALD-GSH accumulation in PMA- activated human neutrophils.
  • Isolated primary human neutrophils (1 x 10 6 ) were incubated in the presence and absence of 200 nM PMA at 37 ° C for indicated time intervals.
  • *** indicates p ⁇ 0.001 for comparisons of each FALD-GSH molecular species at indicated time points compared those at t 0.
  • FIGS. 9A-B AT inhibition of cc-ClFALD and FALD-GSH adduct production in human neutrophils.
  • Isolated primary human neutrophils (1 x 10 6 ) were incubated in the presence and absence of 200 nM PMA as well as the presence and absence of AT at 37 ° C for 30 min.
  • FIGS. 10A-B Mouse Lung/Plasma Analysis - Bromine Exposure.
  • FIG. 11 Mouse Plasma Analysis - Chlorine Exposure.
  • FIG. 12A-B FALD-GSH accumulation in PMA-stimulated eosinophils.
  • FIG. 13 CLP Septic Rat Plasma.
  • FIG. 14 Plasma levels of FALD-GSH and a-ClFA are elevated in K/BxN murine model of arthritis.
  • 2-C1HA and 2-ClOA as well as HDA-GSH and ODA-GSH were quantitated from mouse plasma by LC-MS/MS.
  • n 3 for each treatment. ** and *** indicate p ⁇ 0.01 and 0.001, respectively, for comparisons between C57B1/6J and
  • the disclosure relates to the use of recently discovered adducts formed from the reaction of glutathione with 2-halofatty aldehydes as plasma biomarkers of leukocyte-mediated disease and halogen gas exposure.
  • 2-halofatty aldehydes are produced by activated leukocytes (Thukkani et al, 2003, 2005) as well as during halogen gas exposure.
  • the inventors recently discovered that these aldehydes are chemically modified by nucleophilic attack by glutathione (GSH) leading to glutathione adducts of these aldehydes (FALD-GSH).
  • GSH glutathione
  • FALD-GSH glutathione adducts of these aldehydes
  • FALD-GSH accumulates when leukocytes are activated and are blood borne.
  • FALD-GSH accumulation in blood and or sputum can be an indicator of pro-inflammatory leukocyte mediated disease as well as halogen gas exposure.
  • the inventors have synthesized this adduct as well as a stable isotope labeled adduct, which is essential for the quantification of this adduct.
  • the adduct can be quantified by high performance liquid chromatography with detection by electrospray ionization mass spectrometry using a method that the inventors have developed.
  • Alternative detection techniques for FALD-GSH include using antibodies to this FALD-GSH, which will make detection of these compounds facile using ELISA assays.
  • FALD-GSH is produced in greater quantities when 2-bromofatty aldehyde is the adducted 2-halofatty aldehyde.
  • metabolites include FALD-cysteine-glycine, FALD- cysteine, and FALD-N-acetyl cysteine. These metabolites are logical based on the mercapturate metabolic pathway (Cooper and Hanigan, 2010; FIG. 15).
  • the inventors propose new diagnostic assays for assessing eosinophilic-mediated abnormalities, and possibly for monocyte- and neutrophil- driven disease.
  • the following are exemplary but non-limiting conditions to which the assays may be applied.
  • Asthma is a common chronic inflammatory disease of the airways characterized by variable and recurring symptoms, reversible airflow obstruction and bronchospasm. Common symptoms include wheezing, coughing, chest tightness, and shortness of breath.
  • Asthma is thought to be caused by a combination of genetic and environmental factors. Its diagnosis is usually based on the pattern of symptoms, response to therapy over time and spirometry. It is clinically classified according to the frequency of symptoms, forced expiratory volume in one second (FEV1), and peak expiratory flow rate. Asthma may also be classified as atopic (extrinsic) or non-atopic (intrinsic) where atopy refers to a predisposition toward developing type 1 hypersensitivity reactions.
  • Treatment of acute symptoms is usually with an inhaled short-acting beta-2 agonist (such as salbutamol) and oral corticosteroids.
  • beta-2 agonist such as salbutamol
  • intravenous corticosteroids, magnesium sulfate, and hospitalization may be required.
  • Symptoms can be prevented by avoiding triggers, such as allergens and irritants, and by the use of inhaled corticosteroids.
  • Long-acting beta agonists (LAB A) or antileukotriene agents arachi donate 5 -lipoxygenase inhibitors or CysLTi antagonists
  • LAB A Long-acting beta agonists
  • antileukotriene agents arachi donate 5 -lipoxygenase inhibitors or CysLTi antagonists
  • the occurrence of asthma has increased significantly since the 1970s. In 2011, 235-300 million people globally were diagnosed with asthma, and it caused 250,000 deaths.
  • Asthma is characterized by recurrent episodes of wheezing, shortness of breath, chest tightness, and coughing. Sputum may be produced from the lung by coughing but is often hard to bring up. During recovery from an attack, it may appear pus-like due to high levels of white blood cells called eosinophils. Symptoms are usually worse at night and in the early morning or in response to exercise or cold air. Some people with asthma rarely experience symptoms, usually in response to triggers, whereas others may have marked and persistent symptoms.
  • GFD gastro-esophageal reflux disease
  • rhinosinusitis a number of other health conditions occur more frequently in those with asthma, including gastro-esophageal reflux disease (GERD), rhinosinusitis, and obstructive sleep apnea.
  • GFD gastro-esophageal reflux disease
  • obstructive sleep apnea a number of other health conditions occur more frequently in those with asthma, including gastro-esophageal reflux disease (GERD), rhinosinusitis, and obstructive sleep apnea.
  • Psychological disorders are also more common, with anxiety disorders occurring in between 16-52% and mood disorders in 14-41%.
  • Those with asthma, especially if it is poorly controlled, are at high risk for radiocontrast reactions.
  • Asthma is caused by a combination of complex and incompletely understood environmental and genetic interactions. These factors influence both its severity and its responsiveness to treatment. It is believed that the recent increased rates of asthma are due to changing epigenetics (heritable factors other than those related to the DNA sequence) and a changing living environment.
  • Asthma is associated with exposure to indoor allergens.
  • indoor allergens include: dust mites, cockroaches, animal dander, and mold.
  • Efforts to decrease dust mites have been found to be ineffective.
  • Certain viral respiratory infections, such as respiratory syncytial virus and rhinovirus may increase the risk of developing asthma when acquired as young children.
  • Certain other infections may decrease the risk.
  • Hygiene The hygiene hypothesis attempts to explain the increased rates of asthma worldwide as a direct and unintended result of reduced exposure, during childhood, to nonpathogenic bacteria and viruses. It has been proposed that the reduced exposure to bacteria and viruses is due, in part, to increased cleanliness and decreased family size in modem societies. Exposure to bacterial endotoxin in early childhood may prevent the development of asthma, but exposure at an older age may provoke bronchoconstriction. Evidence supporting the hygiene hypothesis includes lower rates of asthma on farms and in households with pets.
  • Some genetic variants may only cause asthma when they are combined with specific environmental exposures.
  • An example is a specific single nucleotide polymorphism in the CD 14 region and exposure to endotoxin (a bacterial product). Endotoxin exposure can come from several environmental sources including tobacco smoke, dogs, and farms. Risk for asthma, then, is determined by both a person's genetics and the level of endotoxin exposure.
  • a triad of atopic eczema, allergic rhinitis and asthma is called atopy.
  • the strongest risk factor for developing asthma is a history of atopic disease; with asthma occurring at a much greater rate in those who have either eczema or hay fever.
  • Asthma has been associated with Churg-Strauss syndrome, an autoimmune disease and vasculitis. Individuals with certain types of urticaria may also experience symptoms of asthma.
  • Beta blocker medications such as propranolol can trigger asthma in those who are susceptible. Cardioselective beta-blockers, however, appear safe in those with mild or moderate disease. Other medications that can cause problems in same are ASA, NSAIDs, and angiotensin-converting enzyme inhibitors. COX-2 inhibitors do not appear to be a concern.
  • Exacerbation Some individuals will have stable asthma for weeks or months and then suddenly develop an episode of acute asthma. Different individuals react to various factors in different ways. Most individuals can develop severe exacerbation from a number of triggering agents. Some factors that can lead to exacerbation of asthma include dust, animal dander (especially cat and dog hair), cockroach allergens and mold. Perfumes are a common cause of acute attacks in women and children. Both viral and bacterial infections of the upper respiratory tract can worsen the disease. Psychological stress may worsen symptoms— it is thought that stress alters the immune system and thus increases the airway inflammatory response to allergens and irritants.
  • Asthma is the result of chronic inflammation of the airways which subsequently results in increased contractability of the surrounding smooth muscles. This among other factors leads to bouts of narrowing of the airway and the classic symptoms of wheezing. The narrowing is typically reversible with or without treatment. Occasionally the airways themselves change. Typical changes in the airways include an increase in eosinophils and thickening of the lamina reticularis. Chronically the airways' smooth muscle may increase in size along with an increase in the numbers of mucous glands. Other cell types involved include: T lymphocytes, macrophages, and neutrophils. There may also be involvement of other components of the immune system including: cytokines, chemokines, histamine, and leukotrienes among others.
  • asthma While asthma is a well recognized condition, there is not one universal agreed upon definition. It is defined by the Global Initiative for Asthma as "a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. The chronic inflammation is associated with airway hyper-responsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness and coughing particularly at night or in the early morning. These episodes are usually associated with widespread but variable airflow obstruction within the lung that is often reversible either spontaneously or with treatment.”
  • Spirometry Spirometry is recommended to aid in diagnosis and management. It is the single best test for asthma. If the FEV1 measured by this technique improves more than 12% following administration of a bronchodilator such as salbutamol, this is supportive of the diagnosis. It however may be normal in those with a history of mild asthma, not currently acting up. As caffeine is a bronchodilator in people with asthma, the use of caffeine before a lung function test may interfere with the results. Single-breath diffusing capacity can help differentiate asthma from COPD. It is reasonable to perform spirometry every one or two years to follow how well a person's asthma is controlled.
  • the methacholine challenge involves the inhalation of increasing concentrations of a substance that causes airway narrowing in those predisposed. If negative it means that a person does not have asthma; if positive, however, it is not specific for the disease.
  • Other supportive evidence includes: a >20% difference in peak expiratory flow rate on at least three days in a week for at least two weeks, a >20% improvement of peak flow following treatment with either salbutamol, inhaled corticosteroids or prednisone, or a >20% decrease in peak flow following exposure to a trigger. Testing peak expiratory flow is more variable than spirometry, however, and thus not recommended for routine diagnosis. It may be useful for daily self-monitoring in those with moderate to severe disease and for checking the effectiveness of new medications. It may also be helpful in guiding treatment in those with acute exacerbations.
  • Eosinophilic esophagitis is an increasingly recognized eosinophilic gastrointestinal disease (EGID). It accounts for about 50% of dysphagia and food impaction. It has been proposed that food allergy is the underlying etiology of EE. Recent translational studies show that skin prick tests (SPTs) are currently the best available tool to identify the triggering food allergens and >90% of patients respond to dietary interventions, thus supporting a role for humoral (IgE) and/or cell-mediated food allergy. In addition, esophageal mast cells are significantly increased in EE compared to normal controls and gastroesophageal reflux disease (GERD).
  • GID gastroesophageal reflux disease
  • EE is a disorder of the esophagus characterized by esophageal and/or upper gastrointestinal tract symptoms in association with esophageal mucosal biopsy specimens containing high amount of intraepithelial eosinophils within the esophageal squamous epithelium or deeper tissue levels and normal pH monitoring.
  • EE affects males more than females, and the diagnosis is typically made in adults during the third and fourth decades of life, although it may be diagnosed at a later age. In children, the diagnosis is made after infancy and through adolescence with no recognized peak age of onset. Symptoms may include chest pain, heartburn, dysphagia, food impaction and a lack of responsiveness to acid reducing medications. Treatment of EE involves either corticosteroids or elemental diet and not surgery.
  • Standard of care for EE patients includes initial esophageal endoscopy with biopsy to determine the numbers of epithelial eosinophils (>5/hpf being diagnostic). Since consequences of chronic eosinophilic inflammation in EE can include esophageal remodeling with subsequent esophageal narrowing, trachealization and strictures, therapeutic efforts are typically devoted toward inducing clinical as well as histological remission. While overall relatively safe, esophageal endoscopy entails procedural risks, is expensive, time consuming and is limited to procuring a 3 mm sample.
  • an Esophageal String Test or EST such as the Enterotest®.
  • a string-based test first used for detection of Giardia infections can be used in its native form to assess esophageal inflammation at both the protein and mRNA levels and may potentially be used to monitor disease activity.
  • the EST may offer a minimally invasive method to assess the presence of inflammation associated with active disease.
  • EE eosinophilic esophagitis
  • mucosal biopsies from patients with EE demonstrate significantly increased numbers of mucosal mast cells compared to those from patients with gastroesophageal reflux disease (GERD) or normal subjects, suggesting their participation in the pathogenesis of this disease.
  • GFD gastroesophageal reflux disease
  • previous work suggests that patients with eosinophilic gastrointestinal diseases (EGIDs) and food allergy demonstrate increased expression of CD23 on intestinal epithelial cells and in stool samples. While the precise role of CD23 in food allergic responses is not certain, recent studies suggest that the human CD23a isoform participates as a bidirectional transporter of both free IgE and IgE/antigen complexes, and can potentially deliver IgE and its bound allergen across intestinal epithelial cells to induce mast cell activation. Taken together, these observations provide strong evidence supporting a role of food allergic responses (including those mediated by IgE) in the pathogenesis of EE.
  • EE is characterized by symptoms including abdominal pain, regurgitation, feeding intolerance, food impaction and dysphagia.
  • Histologically, esophageal biopsies contain large numbers of intraepithelial eosinophils (> 15 eosinophils/high power field), often with eosinophil microabscesses and luminal layering.
  • eotaxin-3 is the only potential biomarker that has been identified for EE.
  • the obvious problem is that the only method currently available to determine the state of esophageal mucosal inflammation is endoscopy with mucosal biopsy. While this procedure is relatively safe overall, several downsides do exist including the potential complications of conscious sedation or general anesthesia (hypoxia, allergic reactions to the medications, airway compromise) and risks of the procedure itself (esophageal perforation, bleeding and infection). Endoscopy with biopsy is limited in that each biopsy only provides an assessment of ⁇ 0.001 % of the total esophageal surface area. Endoscopy is costly and often is not covered by insurance companies when repeated as described above.
  • Atherosclerosis also known as arteriosclerotic vascular disease or ASVD
  • ASVD arteriosclerotic vascular disease
  • fatty streaks early on because of appearance being similar to that of marbled steak.
  • WBCs white blood cells
  • the remnants eventually include calcium and other crystallized materials, within the outer-most and oldest plaque.
  • the "fatty streaks” reduce the elasticity of the artery walls. However, they do not affect blood flow for decades, because the artery muscular wall enlarges at the locations of plaque. The wall stiffening may eventually increase pulse pressure; widened pulse pressure is one possible result of advanced disease within the major arteries.
  • Atherosclerosis is therefore a syndrome affecting arterial blood vessels due to a chronic inflammatory response of WBCs in the walls of arteries. This is promoted by low-density lipoproteins (LDL, plasma proteins that carry cholesterol and triglycerides) without adequate removal of fats and cholesterol from the macrophages by functional high-density lipoproteins (HDL). It is commonly referred to as a "hardening" or furring of the arteries. It is caused by the formation of multiple atheromatous plaques within the arteries, which are divided into three distinct components:
  • the atheroma which is the nodular accumulation of a soft, flaky, yellowish material at the center of large plaques, composed of macrophages nearest the lumen of the artery
  • Atherosclerosis is a chronic disease that remains asymptomatic for decades. Atherosclerotic lesions, or atherosclerotic plaques, are separated into two broad categories: Stable and unstable (also called vulnerable). The pathobiology of atherosclerotic lesions is very complicated but generally, stable atherosclerotic plaques, which tend to be asymptomatic, are rich in extracellular matrix and smooth muscle cells, while, unstable plaques are rich in macrophages and foam cells and the extracellular matrix separating the lesion from the arterial lumen (also known as the fibrous cap) is usually weak and prone to rupture. Ruptures of the fibrous cap expose thrombogenic material, such as collagen, to the circulation and eventually induce thrombus formation in the lumen.
  • thrombogenic material such as collagen
  • intraluminal thrombi can occlude arteries outright (e.g. coronary occlusion), but more often they detach, move into the circulation and eventually occluding smaller downstream branches causing thromboembolism.
  • coronary occlusion e.g. coronary occlusion
  • atherosclerotic lesions can cause complete closure of the lumen.
  • Chronically expanding lesions are often asymptomatic until lumen stenosis is so severe (usually over 80%) that blood supply to downstream tissue(s) is insufficient, resulting in ischemia.
  • Atherosclerosis affects the entire artery tree, but mostly larger, high-pressure vessels such as the coronary, renal, femoral, cerebral, and carotid arteries. These are termed “clinically silent” because the person having the infarction does not notice the problem and does not seek medical help, or when they do, physicians do not recognize what has happened.
  • Atherosclerosis is initiated by inflammatory processes in the endothelial cells of the vessel wall in response to retained low- density lipoprotein (LDL) particles.
  • LDL low- density lipoprotein
  • LDL particles Lipoproteins in the blood vary in size. Some data suggests that small dense LDL (sdLDL) particles are more prone to pass between the endothelial cells, going behind the cellular monolayer of endothelium. LDL particles and their content are susceptible to oxidation by free radicals, and the risk is higher while the particles are in the wall than while in the bloodstream. However, LDL particles have a half-life of only a couple of days, and their content (LDL particles typically carry 3,000 to 6,000 fat molecules, including: cholesterol, phospholipids, cholesteryl esters, triglycerides & all other fats in the water outside cells, to the tissues of the body) changes with time.
  • LDL particles typically carry 3,000 to 6,000 fat molecules, including: cholesterol, phospholipids, cholesteryl esters, triglycerides & all other fats in the water outside cells, to the tissues of the body
  • Oxidation mechanisms associated with vascular inflammation have been given considerable attention as a mechanism mediating atherosclerotic lesion formation and propagation.
  • Myeloperoxidase (MPO)-catalyzed oxidation is considered to be a major mechanism mediating human atherosclerotic disease.
  • Monocyte- and macrophage-derived MPO catalyzes the production of HOC1 and its conjugate base (OC1 " ).
  • Multiple MPO-derived oxidation products have been found in human atherosclerotic lesions including chlorotyrosine, nitrotyrosine and a-CIFALD.
  • LDL particles can become more prone to oxidation.
  • Endothelial cells respond by attracting monocyte white blood cells, causing them to leave the blood stream, penetrate into the arterial walls and transform into macrophages.
  • the macrophages' ingestion of oxidized LDL particles triggers a cascade of immune responses which over time can produce an atheroma if HDL removal of fats from the macrophages does not keep up.
  • the immune system's specialized white blood cells (macrophages and T- lymphocytes) absorb the oxidized LDL, forming specialized foam cells.
  • foam cells are not able to process the oxidized LDL and recruit HDL particles to remove the fats, they grow and eventually rupture, leaving behind cellular membrane remnants, oxidized materials, and fats (including cholesterol) in the artery wall. This attracts more white blood cells, resulting in a snowballing progression that continues the cycle, inflaming the artery.
  • the presence of the plaque induces the muscle cells of the blood vessel to stretch, compensating for the additional bulk, and the endothelial lining thickens, increasing the separation between the plaque and lumen. This somewhat offsets the narrowing caused by the growth of the plaque, but it causes the wall to stiffen and become less compliant to stretching with each heart beat.
  • Atherosclerosis may be caused by an infection of the vascular smooth muscle cells.
  • Chickens for example, develop atherosclerosis when infected with the Marek's disease herpesvirus.
  • Herpesvirus infection of arterial smooth muscle cells has been shown to cause cholesteryl ester (CE) accumulation, which is associated with atherosclerosis.
  • Cytomegalovirus (CMV) infection is also associated with cardiovascular diseases.
  • Plaques that have ruptured are called complicated plaques.
  • the extracellular matrix of the lesion breaks, usually at the shoulder of the fibrous cap that separates the lesion from the arterial lumen, where the exposed thrombogenic components of the plaque, mainly collagen will trigger thrombus formation.
  • the thrombus then travels downstream to other blood vessels, where the blood clot may partially or completely block blood flow. If the blood flow is completely blocked, cell deaths occur due to the lack of oxygen supply to nearby cells, resulting in necrosis.
  • the narrowing or obstruction of blood flow can occur in any artery within the body. Obstruction of arteries supplying the heart muscle result in a heart attack, while the obstruction of arteries supplying the brain result in a stroke.
  • Lumen stenosis that is greater than 75% were considered as the hallmark of clinically significant disease in the past because recurring episodes of angina and abnormalities in stress test are only detectable at that particular severity of stenosis.
  • clinical trials have shown that only about 14% of clinically debilitating events occur at sites with >75% stenosis.
  • Majority of cardiovascular events that involve sudden rupture of the atheroma plaque do not display any evident narrowing of the lumen.
  • greater attention has been focused on "vulnerable plaque" from the late 1990's onwards.
  • detection approaches include anatomical detection and physiologic measurement.
  • anatomical detection methods include (1) coronary calcium scoring by CT, (2) carotid IMT (intimal media thickness) measurement by ultrasound, and (3) intravascular ultrasound (IVUS).
  • physiologic measurement methods include (1) lipoprotein subclass analysis, (2) HbAlc, (3) hs-CRP, and (4) homocysteine. Both anatomic and physiologic methods allow early detection before symptoms show up, disease staging and tracking of disease progression. Anatomic methods are more expensive and some of them are invasive in nature, such as IVUS. On the other hand, physiologic methods are often less expensive and safer. But they do not quantify the current state of the disease or directly track progression. In the recent years, ways of estimating the severity of atherosclerotic plaques is also made possible with the developments in nuclear imaging techniques such as PET and SPECT.
  • Sepsis is a serious medical condition characterized by a whole-body inflammatory state caused by infection.
  • sepsis has been used interchangeably with septicaemia and septicemia ("blood poisoning").
  • septicemia is considered a subset of sepsis.
  • the inventors have shown (unpublished data) that myeloperoxidase-derived products are increased in a rat model of sepsis, and chlorinated fatty acids are increased in the cecum in the cecal ligation model of rat sepsis.
  • SIRS systemic inflammatory response syndrome
  • the immunological response that causes sepsis is a systemic inflammatory response causing widespread activation of inflammation and coagulation pathways. This may progress to dysfunction of the circulatory system and, even under optimal treatment, may result in the multiple organ dysfunction syndrome and eventually death.
  • Sepsis is considered present if infection is highly suspected or proven and two or more of the following systemic inflammatory response syndrome (SIRS) criteria are met:
  • hyperventilation >20 breaths per minute or, on blood gas, a PaCC less than 32 mm Hg
  • sepsis severe sepsis (sepsis with acute organ dysfunction) and septic shock (sepsis with refractory arterial hypotension).
  • SIRS systemic inflammatory response syndrome criteria
  • patients with SIRS and acute organ dysfunction may be termed "severe SIRS.”
  • Patients are defined as having "severe sepsis” if they have sepsis plus signs of systemic hypoperfusion; either end organ dysfunction or a serum lactate greater than 4 mmol/dL.
  • Patient are defined as having septic shock if they have sepsis plus hypotension after an appropriate fluid bolus (typically 20 ml/kg of crystaloid).
  • the criteria for diagnosing an adult with sepsis do not apply to infants under one month of age. In infants, only the presence of infection plus a "constellation" of signs and symptoms consistent with the systemic response to infection are required for diagnosis.
  • the therapy of sepsis rests on antibiotics, surgical drainage of infected fluid collections, fluid replacement and appropriate support for organ dysfunction. This may include hemodialysis in kidney failure, mechanical ventilation in pulmonary dysfunction, transfusion of blood products, and drug and fluid therapy for circulatory failure. Ensuring adequate nutrition, if necessary by parenteral nutrition, is important during prolonged illness.
  • drotrecogin alfa activate protein C, one of the coagulation factors
  • a patient must have severe sepsis or septic shock with an APACHE II score of 25 or greater and a low risk of bleeding.
  • Low dose hydrocortisone treatment has shown promise for septic shock patients with relative adrenal insufficiency as defined by ACTH stimulation testing.
  • Standard treatment of infants with suspected sepsis consists of supportive care, maintaining fluid status with intravenous fluids, and the combination of a beta-lactam antibiotic (such as ampicillin) with an aminoglycoside such as gentamicin.
  • a beta-lactam antibiotic such as ampicillin
  • an aminoglycoside such as gentamicin.
  • MI Myocardial infarction
  • AMI acute myocardial infarction
  • heart attack is the interruption of blood supply to a part of the heart, causing heart cells to die. This is most commonly due to occlusion (blockage) of a coronary artery following the rupture of a vulnerable atherosclerotic plaque, which is an unstable collection of lipids (fatty acids) and white blood cells (especially macrophages) in the wall of an artery.
  • lipids fatty acids
  • white blood cells especially macrophages
  • the resulting ischemia (restriction in blood supply) and oxygen shortage if left untreated for a sufficient period of time, can cause damage or death (infarction) of heart muscle tissue (myocardium).
  • Heart attacks are the leading cause of death for both men and women worldwide.
  • MI Magnetic resonance Imaging
  • Oxygen, aspirin, and nitroglycerin may be administered. Morphine was classically used if nitroglycerin was not effective; however, it may increase mortality in the setting of NSTEMI. Coronary intervention (PCI) or fibrinolysis are recommended in those with an STEMI. In people who have multiple blockages and who are relatively stable, or in a few emergency cases, bypass surgery may be an option.
  • PCI Coronary intervention
  • fibrinolysis are recommended in those with an STEMI. In people who have multiple blockages and who are relatively stable, or in a few emergency cases, bypass surgery may be an option.
  • Thrombolytic therapy improves survival rates in patients with acute myocardial infarction if administered in a timely fashion in the appropriate group of patients. If PCI capability is not available within 90 minutes, then choice is to administer thrombolytics within 12 hours of onset of symptoms in patients with ST-segment elevation greater than 0.1 mV in 2 or more contiguous ECG leads, new left bundle-branch block (LBBB), or anterior ST depression consistent with posterior infarction.
  • Tissue plasminogen activator (t-PA) is preferred over streptokinase as achieving a higher rate of coronary artery patency; however, the key lies in speed of the delivery.
  • Clopidogrel may be used as an alternative in cases of a resistance or allergy to aspirin (dose of 300 mg), but a higher dose of clopidogrel may have added benefit.
  • Platelet glycoprotein (GP) Ilb/IIIa-receptor antagonist is another therapy in patients with continuing ischemia or with other high-risk features and to patients in whom a percutaneous coronary intervention (PCI) is planned.
  • PCI percutaneous coronary intervention
  • Eptifibatide and tirofiban are approved for this use, and abciximab also can be used for 12-24 hours in patients with unstable angina or NSTEMI in whom a PCI is planned within the next 24 hours.
  • Heparin and other anticoagulant agents have an established role as adjunct agents in patients receiving t-PA, but not in patients receiving streptokinase. Heparin is also indicated in patients undergoing primary angioplasty. Low molecular- weight heparins (LMWHs) have been shown to be superior to UFHs in patients with unstable angina or NSTEMI. Bivalirudin, a direct thrombin inhibitor, has shown promise in STEMI if combined with high-dose clopidogrel.
  • LMWHs Low molecular- weight heparins
  • Nitrates have no apparent impact on mortality rate in patients with ischemic syndromes, but they are useful in symptomatic relief and preload reduction, so much so that all patients with acute myocardial infarction are given nitrates within the first 48 hours of presentation, unless contraindicated (i.e. , in RV infarction).
  • Beta-blockers may reduce the rates of reinfarction and recurrent ischemia, and thus are administered to patients with Mis unless a contraindication is present.
  • ACE inhibitors reduce mortality rates after myocardial infarction and thus are administered as soon as possible as long as no contraindications are and the patient remains stable. ACE inhibitors have the greatest benefit in patients with ventricular dysfunction. Continue ACE inhibitors indefinitely after myocardial infarction. Angiotensin-receptor blockers may be used as an altemative in patients who develop adverse effects, such as a persistent cough, although initial trials need to be confirmed.
  • PCI is the treatment of choice in most patients with STEMI, assuming a door to balloon time of less than 90 minutes.
  • PCI provides greater coronary patency (>96% thrombolysis), lower risk of bleeding, and instant knowledge about the extent of the underlying disease.
  • the choice of primary PCI should be individualized to each patient's presentation and timing.
  • Primary PCI is also the treatment of choice in patients with cardiogenic shock, patients in whom thrombolysis failed, and those with high risk of bleeding or contraindications to thrombolytic therapy.
  • halogens or halogen elements are a group in the periodic table consisting of five chemically related elements: fluorine (F), chlorine (CI), bromine (Br), iodine (I), and astatine (At).
  • the artificially created element 117 may also be a halogen. In the modem IUPAC nomenclature, this group is known as group 17.
  • halogens The group of halogens is the only periodic table group that contains elements in all three familiar states of matter at standard temperature and pressure. All of the halogens form acids when bonded to hydrogen. Most halogens are typically produced from minerals or salts. The middle halogens - chlorine, bromine and iodine - are often used as disinfectants. Organobromides are the most important class of flame retardants.
  • Elemental halogens are generally toxic. The halogens tend to decrease in toxicity towards the heavier halogens.
  • fluorine gas is extremely toxic; breathing fluorine gas at a concentration of 0.1% for several minutes is lethal.
  • Hydrofluoric acid is also toxic, being able to penetrate skin and cause highly painful burns.
  • fluoride anions are toxic, but not as toxic as pure fluorine. Fluoride can be lethal in amounts of 5 to 10 grams. Prolonged consumption of fluoride above concentrations of 1.5 mg/L is associated with a risk of dental fluorosis, an aesthetic condition of the teeth.
  • Chlorine gas is highly toxic. Breathing in chlorine at a concentration of 3 parts per million can rapidly cause a toxic reaction. Breathing in chlorine at a concentration of 50 parts per million is highly dangerous. Breathing in chlorine at a concentration of 500 parts per million for a few minutes is lethal. Breathing in chlorine gas is highly painful. Hydrochloric acid is a dangerous chemical.
  • bromine is somewhat toxic, but less toxic than fluorine and chlorine.
  • Bromide anions are also toxic, but less so than bromine.
  • Bromide has a lethal dose of 30 grams.
  • Iodine is somewhat toxic, being able to irritate the lungs and eyes, with a safety limit of 1 milligram per cubic meter. When taken orally, 3 grams of iodine can be lethal.
  • Iodide anions are mostly nontoxic, but these can also be deadly if ingested in large amounts.
  • Astatine is very radioactive and thus highly dangerous.
  • Antibodies may be generated against protein targets in the glutathione adducts of 2- halofatty aldehydes (FALD-GSH) described herein. Antibodies are defined by their binding specificity. Those of skill in the art are well aware of methods by which such antibodies can be made and identified. Assessing the binding specificity/affinity of a given antibody using techniques is also well known to those of skill in the art, thereby permitting one to determine what antibodies fall within the scope of this disclosure.
  • FLD-GSH 2- halofatty aldehydes
  • Antibody molecules will comprise fragments (such as F(ab'), F(ab')2) that are produced, for example, by the proteolytic cleavage of the mAbs, or single-chain immunoglobulins producible, for example, via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can be combined with one another, or with other antibody fragments or receptor ligands to form "chimeric" binding molecules. Significantly, such chimeric molecules may contain substituents capable of binding to different epitopes of the same molecule.
  • Antibodies may be associated with a label or reporter molecule, which is defined here as any moiety which may be detected using an assay.
  • reporter molecules include enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles or ligands, such as biotin.
  • the labels used can be paramagnetic ions, radioactive isotopes, fluorochromes, NMR-detectable substances, and X-ray imaging agents.
  • paramagnetic ions such as chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and/or erbium (III), with gadolinium being particularly preferred.
  • Ions useful in other contexts, such as X-ray imaging include but are not limited to lanthanum (III), gold (III), lead (II), and especially bismuth (III).
  • radioactive isotopes for therapeutic and/or diagnostic application, one might mention astatine 211 , 14 carbon, 51 chromium, 6 chlorine, "cobalt, 58 cobalt, copper 67 , 152 Eu, gallium 67 , 3 ⁇ 4ydrogen, iodine 123 , iodine 125 , iodine 131 , indium 111 , 59 iron, 2 phosphorus, rhenium 186 , rhenium 188 , 75 selenium, 5 sulphur, technicium 99 " 1 and/or yttrium 90 .
  • Radioactively labeled monoclonal antibodies may be produced according to well-known methods in the art.
  • fluorescent labels contemplated for use as conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6- JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
  • the disclosure provides method for detection and quantitation of FALD- GSH, and the diagnosis of eosinophilic abnormalities based thereon.
  • a variety of different methodologies are available for the detection of FALD-GSH. In general, one can detect either FALD-GSH using antibodies that bind specifically or preferentially to this molecule, or one can employ HPLC and/or mass spectrometric methods. These technologies are described in greater detail below.
  • MS mass spectrometry
  • mass spectrometry may be used to look for the levels of these proteins particularly.
  • ESI ESI is a convenient ionization technique developed by Fenn and colleagues (Fenn et al , 1989) that is used to produce gaseous ions from highly polar, mostly nonvolatile biomolecules, including lipids.
  • the sample is injected as a liquid at low flow rates (1-10 ⁇ /min) through a capillary tube to which a strong electric field is applied.
  • the field generates additional charges to the liquid at the end of the capillary and produces a fine spray of highly charged droplets that are electrostatically attracted to the mass spectrometer inlet.
  • the evaporation of the solvent from the surface of a droplet as it travels through the desolvation chamber increases its charge density substantially. When this increase exceeds the Rayleigh stability limit, ions are ejected and ready for MS analysis.
  • a typical conventional ESI source consists of a metal capillary of typically 0.1-0.3 mm in diameter, with a tip held approximately 0.5 to 5 cm (but more usually 1 to 3 cm) away from an electrically grounded circular interface having at its center the sampling orifice, such as described by Kabarle et al (1993).
  • a potential difference of between 1 to 5 kV (but more typically 2 to 3 kV) is applied to the capillary by power supply to generate a high electrostatic field (10 6 to 10 7 V/m) at the capillary tip.
  • a sample liquid carrying the analyte to be analyzed by the mass spectrometer is delivered to tip through an internal passage from a suitable source (such as from a chromatograph or directly from a sample solution via a liquid flow controller).
  • a suitable source such as from a chromatograph or directly from a sample solution via a liquid flow controller.
  • the liquid leaves the capillary tip as small highly electrically charged droplets and further undergoes desolvation and breakdown to form single or multicharged gas phase ions in the form of an ion beam.
  • the ions are then collected by the grounded (or negatively charged) interface plate and led through an the orifice into an analyzer of the mass spectrometer. During this operation, the voltage applied to the capillary is held constant.
  • ESI tandem mass spectroscopy In ESI tandem mass spectroscopy (ESI/MS/MS), one is able to simultaneously analyze both precursor ions and product ions, thereby monitoring a single precursor product reaction and producing (through selective reaction monitoring (SRM)) a signal only when the desired precursor ion is present.
  • SRM selective reaction monitoring
  • the internal standard is a stable isotope-labeled version of the analyte, this is known as quantification by the stable isotope dilution method.
  • This approach has been used to accurately measure pharmaceuticals (Zweigenbaum et al , 2000; Zweigenbaum et al, 1999) and bioactive peptides (Desiderio et al, 1996; Lovelace et al , 1991).
  • Newer methods are performed on widely available MALDI-TOF instruments, which can resolve a wider mass range and have been used to quantify metabolites, peptides, and proteins.
  • Larger molecules such as peptides can be quantified using unlabeled homologous peptides as long as their chemistry is similar to the analyte peptide (Duncan et al , 1993; Bucknall et al, 2002). Protein quantification has been achieved by quantifying tryptic peptides (Mirgorodskaya et al , 2000). Complex mixtures such as crude extracts can be analyzed, but in some instances sample clean up is required (Nelson et al , 1994; Gobom ei al, 2000).
  • Secondary ion mass spectroscopy is an analytical method that uses ionized particles emitted from a surface for mass spectroscopy at a sensitivity of detection of a few parts per billion.
  • the sample, surface is bombarded by primary energetic particles, such as electrons, ions (e.g. , O, Cs), neutrals or even photons, forcing atomic and molecular particles to be ejected from the surface, a process called sputtering. Since some of these sputtered particles carry a charge, a mass spectrometer can be used to measure their mass and charge. Continued sputtering permits measuring of the exposed elements as material is removed. This in turn permits one to construct elemental depth profiles. Although the majority of secondary ionized particles are electrons, it is the secondary ions which are detected and analysis by the mass spectrometer in this method.
  • Laser desorption mass spectroscopy involves the use of a pulsed laser, which induces desorption of sample material from a sample site-effectively, this means vaporization of sample off of the sample substrate. This method is usually only used in conjunction with a mass spectrometer, and can be performed simultaneously with ionization if one uses the right laser radiation wavelength.
  • LD-MS When coupled with Time-of-Flight (TOF) measurement, LD-MS is referred to as LDLPMS (Laser Desorption Laser Photoionization Mass Spectroscopy).
  • LDLPMS Laser Desorption Laser Photoionization Mass Spectroscopy
  • the LDLPMS method of analysis gives instantaneous volatilization of the sample, and this form of sample fragmentation permits rapid analysis without any wet extraction chemistry.
  • the LDLPMS instrumentation provides a profile of the species present while the retention time is low and the sample size is small.
  • an impactor strip is loaded into a vacuum chamber. The pulsed laser is fired upon a certain spot of the sample site, and species present are desorbed and ionized by the laser radiation. This ionization also causes the molecules to break up into smaller fragment-ions.
  • the positive or negative ions made are then accelerated into the flight tube, being detected at the end by a microchannel plate detector.
  • Signal intensity, or peak height, is measured as a function of travel time.
  • the applied voltage and charge of the particular ion determines the kinetic energy, with separation of fragments due to different size causing different velocity. Each ion mass will thus have a different flight-time to the detector.
  • Positive ions are made from regular direct photoionization, but negative ion formation requires a higher powered laser and a secondary process to gain electrons. Most of the molecules that come off the sample site are neutrals, and thus can attract electrons based on their electron affinity. The negative ion formation process is less efficient than forming just positive ions. The sample constituents will also affect the outlook of a negative ion spectra.
  • MALDI-TOF-MS Since its inception and commercial availability, the versatility of MALDI-TOF-MS has been demonstrated convincingly by its extensive use for qualitative analysis. For example, MALDI-TOF-MS has been employed for the characterization of synthetic polymers (Marie et al , 2000; Wu et al, 1998). peptide and protein analysis (Roepstorff et al , 2000; Nguyen et al , 1995), DNA and oligonucleotide sequencing (Miketova et al , 1997; Faulstich et al , 1997; Bentzley et al , 1996), and the characterization of recombinant proteins (Kanazawa et al.
  • MALDI-TOF-MS The properties that make MALDI-TOF-MS a popular qualitative tool—its ability to analyze molecules across an extensive mass range, high sensitivity, minimal sample preparation and rapid analysis times—also make it a potentially useful quantitative tool.
  • MALDI-TOF-MS also enables non-volatile and thermally labile molecules to be analyzed with relative ease. It is therefore prudent to explore the potential of MALDI-TOF-MS for quantitative analysis in clinical settings, for toxicological screenings, as well as for environmental analysis.
  • the application of MALDI-TOF-MS to the quantification of peptides and proteins is particularly relevant. The ability to quantify intact proteins in biological tissue and fluids presents a particular challenge in the expanding area of proteomics and investigators urgently require methods to accurately measure the absolute quantity of proteins.
  • the properties of the matrix material used in the MALDI method are critical. Only a select group of compounds is useful for the selective desorption of proteins and polypeptides. A review of all the matrix materials available for peptides and proteins shows that there are certain characteristics the compounds must share to be analytically useful. Despite its importance, very little is known about what makes a matrix material "successful" for MALDI. The few materials that do work well are used heavily by all MALDI practitioners and new molecules are constantly being evaluated as potential matrix candidates. With a few exceptions, most of the matrix materials used are solid organic acids. Liquid matrices have also been investigated, but are not used routinely.
  • High-performance liquid chromatography is a technique in analytic chemistry used to separate the components in a mixture, to identify each component, and to quantify each component. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causing different flow rates for the different components and leading to the separation of the components as they flow out the column.
  • HPLC has been used for medical (e.g. , detecting vitamin D levels in blood serum), legal (e.g. , detecting performance enhancement drugs in urine), research (e.g., separating the components of a complex biological sample, or of similar synthetic chemicals from each other), and manufacturing (e.g. , during the production process of pharmaceutical and biological products) purposes.
  • medical e.g. , detecting vitamin D levels in blood serum
  • legal e.g. , detecting performance enhancement drugs in urine
  • research e.g., separating the components of a complex biological sample, or of similar synthetic chemicals from each other
  • manufacturing e.g. , during the production process of pharmaceutical and biological products
  • HPLC Chromatography can be described as a mass transfer process involving adsorption.
  • HPLC relies on pumps to pass a pressurized liquid and a sample mixture through a column filled with a sorbent, leading to the separation of the sample components.
  • the active component of the column, the sorbent is typically a granular material made of solid particles (e.g. , silica, polymers, etc.), 2-50 micrometers in size.
  • the components of the sample mixture are separated from each other due to their different degrees of interaction with the sorbent particles.
  • the pressurized liquid is typically a mixture of solvents (e.g.
  • composition and temperature play a major role in the separation process by influencing the interactions taking place between sample components and sorbent. These interactions are physical in nature, such as hydrophobic (dispersive), dipole-dipole and ionic, most often a combination thereof.
  • HPLC is distinguished from traditional (“low pressure") liquid chromatography because operational pressures are significantly higher (50-350 bar), while ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. Due to the small sample amount separated in analytical HPLC, typical column dimensions are 2.1-4.6 mm diameter, and 30-250 mm length. Also HPLC columns are made with smaller sorbent particles (2-50 micrometer in average particle size). This gives HPLC superior resolving power when separating mixtures, which is why it is a popular chromatographic technique.
  • An HPLC instrument typically includes a sampler, pumps, and a detector.
  • the sampler brings the sample mixture into the mobile phase stream which carries it into the column.
  • the pumps deliver the desired flow and composition of the mobile phase through the column.
  • the detector generates a signal proportional to the amount of sample component emerging from the column, hence allowing for quantitative analysis of the sample components.
  • a digital microprocessor and user software control the HPLC instrument and provide data analysis.
  • Some models of mechanical pumps in a HPLC instrument can mix multiple solvents together in ratios changing in time, generating a composition gradient in the mobile phase.
  • Various detectors are in common use, such as UV/Vis, photodiode array (PDA) or based on mass spectrometry.
  • Most HPLC instruments also have a column oven that allows for adjusting the temperature the separation is performed at.
  • immunodetection methods for identifying and/or quantifying FALD-GSH.
  • Some immunodetection methods include enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometric assay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay, and Western blot to mention a few.
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • immunoradiometric assay immunoradiometric assay
  • fluoroimmunoassay chemiluminescent assay
  • bioluminescent assay bioluminescent assay
  • Western blot to mention a few.
  • the steps of various useful immunodetection methods have been described in the scientific literature, such as, e.g. , Doolittle and Ben-Zeev (1999), Gulbis and Galand£1993), De Jager et al. (1993), and Nakamura et
  • the immunobinding methods include obtaining a sample and contacting the sample with a first antibody in accordance with embodiments discussed herein, as the case may be, under conditions effective to allow the formation of immunocomplexes. It is also possible to perform in vivo assays.
  • the antibody employed in the detection may itself be linked to a detectable label, wherein one would then simply detect this label, thereby allowing the amount of the primary immune complexes in the composition to be determined.
  • the first antibody that becomes bound within the primary immune complexes may be detected by means of a second binding ligand that has binding affinity for the antibody.
  • the second binding ligand may be linked to a detectable label.
  • the second binding ligand is itself often an antibody, which may thus be termed a "secondary" antibody.
  • the primary immune complexes are contacted with the labeled, secondary binding ligand, or antibody, under effective conditions and for a period of time sufficient to allow the formation of secondary immune complexes.
  • the secondary immune complexes are then generally washed to remove any non-specifically bound labeled secondary antibodies or ligands, and the remaining label in the secondary immune complexes is then detected.
  • Further methods include the detection of primary immune complexes by a two step approach.
  • a second binding ligand such as an antibody that has binding affinity for the antibody, is used to form secondary immune complexes, as described above.
  • the secondary immune complexes are contacted with a third binding ligand or antibody that has binding affinity for the second antibody, again under effective conditions and for a period of time sufficient to allow the formation of immune complexes (tertiary immune complexes).
  • the third ligand or antibody is linked to a detectable label, allowing detection of the tertiary immune complexes thus formed. This system may provide for signal amplification if this is desired.
  • One method of immunodetection uses two different antibodies.
  • a first biotinylated antibody is used to detect the target antigen, and a second antibody is then used to detect the biotin attached to the complexed biotin.
  • the sample to be tested is first incubated in a solution containing the first step antibody. If the target antigen is present, some of the antibody binds to the antigen to form a biotinylated antibody/antigen complex.
  • the antibody/antigen complex is then amplified by incubation in successive solutions of streptavidin (or avidin), biotinylated DNA, and/or complementary biotinylated DNA, with each step adding additional biotin sites to the antibody/antigen complex.
  • the amplification steps are repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the second step antibody against biotin.
  • This second step antibody is labeled, as for example with an enzyme that can be used to detect the presence of the antibody/antigen complex by histoenzymology using a chromogen substrate.
  • a conjugate can be produced which is macroscopically visible.
  • PCR Polymerase Chain Reaction
  • the PCR method is similar to the Cantor method up to the incubation with biotinylated DNA, however, instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA/biotin/streptavidin/antibody complex is washed out with a low pH or high salt buffer that releases the antibody. The resulting wash solution is then used to carry out a PCR reaction with suitable primers with appropriate controls.
  • the enormous amplification capability and specificity of PCR can be utilized to detect a single antigen molecule.
  • Immunoassays are, in their most simple and direct sense, binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily appreciated that detection is not limited to such techniques, and western blotting, dot blotting, FACS analyses, and the like may also be used.
  • the antibodies of the invention are immobilized onto a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. Then, a test composition suspected of containing the foci is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen may be detected. Detection may be achieved by the addition of another anti-foci antibody that is linked to a detectable label.
  • ELISA is a simple "sandwich ELISA.” Detection may also be achieved by the addition of a second anti-foci antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.
  • the samples suspected of containing the foci are immobilized onto the well surface and then contacted with anti-foci antibody. After binding and washing to remove non-specifically bound immune complexes, the bound anti-foci antibodies are detected. Where the initial anti-foci antibodies are linked to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody that has binding affinity for the first anti-foci antibody, with the second antibody being linked to a detectable label.
  • ELISAs have certain features in common, such as coating, incubating and binding, washing to remove non-specifically bound species, and detecting the bound immune complexes. These are described below.
  • a plate with either antigen or antibody In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein or solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.
  • a secondary or tertiary detection means rather than a direct procedure.
  • the immobilizing surface is contacted with the biological sample to be tested under conditions effective to allow immune complex (antigen/antibody) formation. Detection of the immune complex then requires a labeled secondary binding ligand or antibody, and a secondary binding ligand or antibody in conjunction with a labeled tertiary antibody or a third binding ligand.
  • Under conditions effective to allow immune complex (antigen/antibody) formation means that the conditions preferably include diluting the antigens and/or antibodies with solutions such as BSA, bovine gamma globulin (BGG) or phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of nonspecific background.
  • the "suitable" conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 hours or so, at temperatures preferably on the order of 25 °C to 27 °C, or may be overnight at about 4 °C or so. Following all incubation steps in an ELISA, the contacted surface is washed so as to remove non-complexed material. A preferred washing procedure includes washing with a solution such as PBS/Tween, or borate buffer. Following the formation of specific immune complexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minute amounts of immune complexes may be determined.
  • a solution such as PBS/Tween, or borate buffer.
  • the second or third antibody will have an associated label to allow detection.
  • this will be an enzyme that will generate color development upon incubating with an appropriate chromogenic substrate.
  • a urease, glucose oxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibody for a period of time and under conditions that favor the development of further immune complex formation (e.g. , incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).
  • the amount of label is quantified, e.g. , by incubation with a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6- sulfonic acid (ABTS), or H2O2, in the case of peroxidase as the enzyme label. Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.
  • a chromogenic substrate such as urea, or bromocresol purple, or 2,2'-azino-di-(3-ethyl-benzthiazoline-6- sulfonic acid (ABTS), or H2O2
  • Quantification is then achieved by measuring the degree of color generated, e.g., using a visible spectra spectrophotometer.
  • the Western blot is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel electrophoresis to separate native or denatured proteins by the length of the polypeptide (denaturing conditions) or by the 3-D structure of the protein (native/non-denaturing conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.
  • a membrane typically nitrocellulose or PVDF
  • Samples may be taken from whole tissue or from cell culture. In most cases, solid tissues are first broken down mechanically using a blender (for larger sample volumes), using a homogenizer (smaller volumes), or by sonication. Cells may also be broken open by one of the above mechanical methods. However, it should be noted that bacteria, virus or environmental samples can be the source of protein and thus Western blotting is not restricted to cellular studies only. Assorted detergents, salts, and buffers may be employed to encourage lysis of cells and to solubilize proteins. Protease and phosphatase inhibitors are often added to prevent the digestion of the sample by its own enzymes. Tissue preparation is often done at cold temperatures to avoid protein denaturing.
  • the proteins of the sample are separated using gel electrophoresis. Separation of proteins may be by isoelectric point (pi), molecular weight, electric charge, or a combination of these factors. The nature of the separation depends on the treatment of the sample and the nature of the gel. This is a very useful way to determine a protein. It is also possible to use a two-dimensional (2-D) gel which spreads the proteins from a single sample out in two dimensions. Proteins are separated according to isoelectric point (pH at which they have neutral net charge) in the first dimension, and according to their molecular weight in the second dimension.
  • isoelectric point pH at which they have neutral net charge
  • the proteins In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF).
  • PVDF polyvinylidene difluoride
  • the membrane is placed on top of the gel, and a stack of filter papers placed on top of that. The entire stack is placed in a buffer solution which moves up the paper by capillary action, bringing the proteins with it.
  • Another method for transferring the proteins is called electroblotting and uses an electric current to pull proteins from the gel into the PVDF or nitrocellulose membrane.
  • the proteins move from within the gel onto the membrane while maintaining the organization they had within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below).
  • Both varieties of membrane are chosen for their nonspecific protein binding properties (i.e., binds all proteins equally well). Protein binding is based upon hydrophobic interactions, as well as charged interactions between the membrane and protein. Nitrocellulose membranes are cheaper than PVDF, but are far more fragile and do not stand up well to repeated probings. The uniformity and overall effectiveness of transfer of protein from the gel to the membrane can be checked by staining the membrane with Coomassie Brilliant Blue or Ponceau S dyes. Once transferred, proteins are detected using labeled primary antibodies, or unlabeled primary antibodies followed by indirect detection using labeled protein A or secondary labeled antibodies binding to the Fc region of the primary antibodies.
  • the antibodies may also be used in conjunction with both fresh-frozen and/or formalin- fixed, paraffin-embedded tissue blocks prepared for study by immunohistochemistry (IHC).
  • IHC immunohistochemistry
  • frozen-sections may be prepared by rehydrating 50 ng of frozen "pulverized” tissue at room temperature in phosphate buffered saline (PBS) in small plastic capsules; pelleting the particles by centrifugation; resuspending them in a viscous embedding medium (OCT); inverting the capsule and/or pelleting again by centrifugation; snap-freezing in -70°C isopentane; cutting the plastic capsule and/or removing the frozen cylinder of tissue; securing the tissue cylinder on a cryostat microtome chuck; and/or cutting 25-50 serial sections from the capsule.
  • whole frozen tissue samples may be used for serial section cuttings.
  • Permanent-sections may be prepared by a similar method involving rehydration of the 50 mg sample in a plastic microfuge tube; pelleting; resuspending in 10% formalin for 4 hours fixation; washing/pelleting; resuspending in warm 2.5% agar; pelleting; cooling in ice water to harden the agar; removing the tissue/agar block from the tube; infiltrating and/or embedding the block in paraffin; and/or cutting up to 50 serial permanent sections. Again, whole tissue samples may be substituted.
  • compositions described herein may be comprised in a kit.
  • an antibody targeting FALD-GSH may be included in a kit.
  • the kit may further include a sterile buffer to facilitate dilution.
  • the antibody may be labeled, such as with a fluorescent label.
  • kits will thus comprise, in suitable container means, a first antibody that binds to FALD-GSH, and optionally an immunodetection reagent.
  • the antibody may be pre-bound to a solid support, such as a column matrix and/or well of a microtitre plate.
  • the antibodies may have detectable labels that are associated with or linked to the given antibody. Detectable labels that are associated with or attached to a secondary binding ligand are also contemplated. Exemplary secondary ligands are those secondary antibodies that have binding affinity for the first antibody.
  • the kits may further comprise a suitably aliquoted composition of FALD-GSH, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay.
  • kits may contain antibody-label conjugates either in fully conjugated form, in the form of intermediates, or as separate moieties to be conjugated by the user of the kit.
  • the components of the kits may be packaged either in aqueous media or in lyophilized form.
  • kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
  • the kits of the present disclosure also will typically include a means for containing the antibodies and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Instructions may include variations that can be implemented.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the active agent may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • kits may also include components that preserve or maintain the active agent that protect against its degradation.
  • HPLC grade methanol, acetonitrile, and isopropanol were purchased from Fisher Scientific.
  • TLC Plates (20 x 20cm, 4 ⁇ silica gel) were obtained from EMB Millipore.
  • hexadecanoic acid was purchased from Medical Isotopes. All other chemicals were purchased from Sigma- Aldrich or Fisher Scientific.
  • Electrosprav Mass Spectrometry Characterization TLC purified HDA-GSH was diluted 2000x with ACN/H 2 0 (7:3) with 0.1% formic acid and directly injected onto the Thermo Fisher TSQ Quantum Ultra mass spectrometer at a flow rate of 5 ⁇ /min.
  • electrospray ionization MS Ql
  • the ionization energy and temperature were 3700V and 270 °C respectively for positive ion mode and 2600V and 270 °C for negative ion mode.
  • Further structural data was gathered through Q2 fragmentation with a collision energy of 15eV and collision gas of 1.0 Torr argon for both the positive and negative ions and analysis of the Q3 fragments.
  • the white solid was sequentially washed with 3 x 10 ml Hexane, 2 x 10 ml MEOH, and 2 x 10 ml H 2 0 to remove any remaining free 2-BrHDA and GSH.
  • the final product was brought to dryness by lyophilization and weighed.
  • the resulting product was analyzed by direction injection ESI-MS/MS and found to be pure /£3 ⁇ 4/-HDA-GSH.
  • RAW 264.7 Cell 2-ClHDA Treatments RAW 264.7 cells were treated with Dubelco's Modified Eagle's Medium (DMEM) supplemented with 2% Fetal Bovine Serum (FBS) and 0, 1, 5, 25, or 50 ⁇ 2-ClHDA for 8 hours. The media was then removed and cells washed with PBS. The cells were scraped in 1.25 ml PBS containing 10 mM N-ethylmaleimide (NEM) and immediately frozen. 10 mM NEM was added to the final cell homogenate solution to block any free GSH sulfhydryls from spontaneously reacting with a-chlorofatty aldehydes (a- C IF AID) in following extraction procedures although no spontaneous production has been observed.
  • DMEM Dubelco's Modified Eagle's Medium
  • FBS Fetal Bovine Serum
  • 2-ClHDA 2% Fetal Bovine Serum
  • PMA stimulated Neutrophils Primary human neutrophils were isolated as previously published (1). Briefly, enriched neutrophils were diluted to 1 million neutrophils per ml in Hanks buffer solution. 200 nM phorbol-12-myristate 13-acetate (PMA) in EtOH (0.1%) and 10 mM aminotriazole were then added to respective experiments. The solutions were allowed to incubate for 30 min at 37 °C. At the end of each reaction, lOmM NEM was added and the neutrophils were immediately frozen.
  • PMA phorbol-12-myristate 13-acetate
  • the supernatant was loaded on a Strata-X (60 mg bed weight) conditioned with 1.2 ml MEOH followed by 1.2 ml H 2 0/MEOH (4/1 v/v). Following the load, the column was washed with 2 x 0.6 ml H 2 0/MEOH (4/1 v/v). 2-ClHDA conjugates of GSH were eluted with 2 x 0.6 ml of MEOH/ACN (3/1 v/v) with 0.25% formic acid. The eluate was dried under N2 and suspended in 100 ⁇ 6:4:5 ACN:IPA:H 2 0 (0.15% formic acid) for analysis by LC-MS/MS.
  • a Phenomenex Onyx Monolithic (50 x 2.0mm) column was coupled to the ESI-MS/MS to allow for precise quantitation of the GSH adducts using single reaction monitoring (SRMs).
  • Solvent A was ACN/IPA (3/2 v/v) with 0.25% formic acid and B was H 2 0 (0.25% formic acid).
  • the column was equilibrated with 65/35 A/B followed by the injection 25 ⁇ of the Strata-X purified solutions onto the column.
  • GSH adducts of 2-ClHDA were eluted by a 2.5 min gradient of 35 to 100%B.
  • 2-Chlorohexadecanal Forms in vitro Adducts with Glutathione The alpha carbon of 2-chlorohexadecanal (2-ClHDA) bound by chlorine may be a target for modification by cellular nucleophiles.
  • Scheme 1 demonstrates the structure of 2-ClHDA and its electrophilic target carbons that were theorized to be available for modification.
  • Glutathione is a ubiquitous cellular nucleophile that is known to bind electrophilic lipids and is a likely candidate for cellular modification of a-ClFAlD.
  • FIGS. 1 A-B demonstrate that a new, more lipophilic compound that runs faster on a TLC plate (Rf « 2.4) than GSH (Rf « 0.9) is resolved by silica TLC chromatography (lane 3) with a polar lipid solvent system.
  • the new compound is stained by Ninhydrin (FIG. 1A), which readily stains the amine of GSH, but does not stain 2-ClHDA. Additionally, the new compound is also stained by DNPH (FIG.
  • FIG. 2A demonstrates that pure 2-ClHDA-GSH adduct was attained by extraction (lane 2).
  • the TLC purified band was then diluted and analyzed by direct injection on a Thermo Fisher TSQ Quantum Ultra mass spectrometer.
  • the predominating [M+H] + ion was 546.36 (FIG. 2B).
  • the proposed structure for this compound (FIG.
  • 2D based on a molecular weight of 545.36 has the GSH thiol substituting the chlorine at the alpha position of 2-ClHDA to form a dechlorinated HDA-GSH adduct.
  • Additional ions found in the positive scan correlate with the common addition of Na (+22, 568.32) and loss of H 2 0 (-18, 528.30).
  • the fourth ion found in the positive ion survey scan, 399.30, is a common fragment of GSH adducts of lipids that corresponds to the loss of pyroglutamate and H 2 0 from the parent ion, 546.36.
  • FIG. 2C displays the negative ion survey scan of the TLC purified HDA-GSH, which complements the positive ion scan.
  • the prevailing ion is the [M-H] " , 544.66, which supports the proposed HDA-GSH structure.
  • both the [M+H] + and [M-H] " ions were subjected to MS/MS fragmentation and analysis.
  • the positive parent ion, 546.36 [M+H] + fragments into two major ions, 399.41 and 296.2 (FIG. 3A).
  • the 399.41 fragment corresponds with the loss of pyroglutamate and H 2 0 as previously stated and the 296.32 ion also losses the glycine moiety of GSH and the carbonyl of cysteine in addition to the loss of pyroglutamate and H 2 0.
  • Additional experiments examined the reactivity of cysteine with 2-ClHDA and revealed a readily formed adduct with 296.32 as the primary ion formed (data not shown).
  • the negative parent ion, 544.66 [M-H] " fragments into multiple ions (FIG. 3B) that directly correspond to the known fragmentation of GSH.
  • the predominating ion, 272.16 is the cleavage of HDA-GSH at sulfhydryl as demonstrated in the FIG. 3B inset. Both the positive and negative ion survey scan and parent ion fragmentations support the structure proposed in FIG. 2D.
  • FIG. 4A shows the expected [M+H] + shift from 546.29 to the DNPH derivative at 726.19.
  • FIG 4B displays the fragmentation pattern of the DNPH derivative displayed in FIG 4C.
  • the DNPH derivative verifies the HDA-GSH stricture containing a free aldehyde proposed in FIG 2.
  • FIG. 6A shows a chromatogram using the elution gradient described in "Material and Methods" with an elution time of the natural and deuterated HDA-GSH elute at the same time (Rt 7.82 min).
  • HDA-GSH produced in RAW 264.7 cells can be directly attributed to the exogenous 2-ClHDA.
  • HDA-GSH was first purified out of the cell homogenate using Strata-X columns as described in the methods. No HDA-GSH was detected in the EtOH controls while HDA-GSH levels were elevated in a concentration dependent manor between 10-50 ⁇ 2-ClHDA treatments (FIG. 7).
  • FIG. 8A shows a time course of a-CIFAlD production in 200 nM PMA stimulated neutrophils.
  • Both the 16-carbon 2-chlorohexadecanal (2-ClHDA) and 18-carbon 2- chlorooctadecanal (2-ClODA) a-CIFAlD molecular species were elevated by 5 min, peak by 30 min and begin to decline by 60 min.
  • a-CIFAlD production can be abolished in primary human neutrophils with the addition of aminotriazole.
  • 2-ClHDA and 2-ClODA levels were greatly reduced in 200 nM PMA treated neutrophils at 30 min (FIG. 8A).
  • HDA-GSH and ODA-GSH levels were significantly reduced in 200 nM PMA treated neutrophils at 30 min as well (FIG. 8B).
  • Inhibition of a-CIFAlD and their proposed adducts of GSH (HDA-GSH and ODA-GSH) by aminotriazole demonstrate that the a-CIFAlD are likely the primary, if not only, source of HDA-GSH and ODA-GSH in human neutrophils.
  • FALD-GSH is a biomarker of bromine and chlorine gas exposures.
  • FALD-GSH adducts are made as a result of a-halofatty aldehydes reacting with glutathione (GSH).
  • GSH glutathione
  • the a- halofatty aldehydes that the inventors have previously identified are a-chlorofatty aldehydes and a-bromofatty aldehydes, which are produced as a result of either hypochlorous acid and hypobromous acids, respectively targeting plasmalogens. They have also shown that the halogen gases, chlorine and bromine also react similarly with plasmalogens yielding these a- halofatty aldehydes. Accordingly, they are interested in the production of a-halofatty aldehydes and related compounds as biomarkers of halogen gas exposure.
  • FIG. 10B shows robust increases in FALD-GSH in the plasma of mice following bromine gas exposure while FIG 10A shows the FALD-GSH elvations in the lung. No adduct is observed in the plasma of mice exposed to air only (the first bar). Seventy -two hr following exposure, the amount of adduct in the plasma is still detectable but has decreased indicating that the FALD-GSH is metabolically cleared over time. By Ninety- six hours very little FALD-GSH is detectable.
  • Eosinophils isolated from allergen-hypersensitive individuals were suspended in Hanks' balanced salt solution containing 100 micromolar NaBr at a concentration of 10 6 cells/ml for 1 h under the indicated conditions (with and without 200 nM PMA and with and without the eosinophil peroxidase activity inhibitor, sodium azide (NaAZ). Duplicates of each condition (1 and 2 in FIG. 12) were independently incubated. Following incubations, cell suspensions were snap frozen, and subsequently FALD-GSH was quantified using [A]-HDA- GSH as internal standard and the LC/MS/MS method described herein. The data show the eosinophil peroxidase-dependent production of HDA-GSH and ODA-GSH in PMA-stimulated eosinophils.
  • FIG. 12B it should be appreciated that levels of a-halofatty aldehydes are 20-fold lower in eosinophils compared to neutrophils (FIG. 8A) yet similar levels of FALD-GSH are produced (FIG 12 A. This is because Eosinophils produce primarily 2-Br-FALD, which reacts much faster with glutathione than 2-Cl-FALD produced by neutrophils.
  • FALD-GSH is a biomarker for sepsis and arthritis. Both sepsis and arthritis induce leukocyte activation and the production of FALD-GSH is expected.
  • CLP cecal ligation puncture
  • FALD-GSH was found to be elevated relative to sham control rats. Additionally, FALD-GSH is increased in ten-week old K/BxN mice with inflammation-induced arthritis compared to C57B1/6J control mice (FIG. 14). Both models display elevations of FALD-GSH in the plasma due to leukocyte activation through inflammatory pathologies.
  • FALD-GSH is a different biomarker for reactions in the body that are mediated by hypohalous acids or halogens.
  • FALD-GSH differs by its production through a different metabolic pathway starting with targeting a different biomolecule, which then yields the biomarker (e.g. , tyrosine versus plasmalogen for the FALD-GSH).
  • biomarker e.g. , tyrosine versus plasmalogen for the FALD-GSH.
  • 2-chlorofatty acids FALD-GSH differs in that for this biomarker 2-halofatty aldehyde reacts with glutathione to yield the adducts described in this disclosure rather than being oxidized to yield the 2-halofatty acid (e.g. , 2- chlorofatty acid).
  • FALD-GSH adducts are stable over a longer period of time compared to halotyrosine.
  • One additional difference with FALD-GSH compared to 2-chlorofatty acid is that FALD-GSH is a peptidoaldehyde that can be used as an antigen to develop antibodies for detection.
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods, and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

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Abstract

L'invention concerne des composés d'addition de glutathione d'aldéhydes gras (FALD-GSH) et des procédés utiles dans la détection de FALD-GSH dans l'identification de pathologies associées à des états pathologiques à médiation leucocytaire, y compris l'activation des éosinophiles et des neutrophiles. Ainsi, la présente invention présente des méthodes de diagnostic d'un sujet ayant ou étant à risque de développer une maladie à médiation leucocytaire (LMD), comprenant (a) la détection du niveau de composés d'addition de glutathione d'aldéhydes 2-halofatty (FALD-GSH) dans un échantillon; (b) la comparaison de la quantité de FALD-GSH avec un niveau témoin ou un niveau standard de FALD-GSH sain et/ou malade; et (c) le diagnostic du sujet comme ayant ou étant à risque de développer une LMD si le niveau de FALD-GSH dans l'échantillon est plus élevé que le témoin ou standard.
PCT/US2016/013834 2015-01-21 2016-01-19 Diagnostic de maladie à médiation leucocytaire et exposition à un gaz halogène WO2016118468A1 (fr)

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Citations (2)

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US20110091980A1 (en) * 2009-10-16 2011-04-21 Saint Louis University Diagnostic method for biomarkers of adverse coronary events
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FORD, D.: "Lipid oxidation by hypochlorous acid: chlorinated lipids in atherosclerosis and myocardial ischemia", CLIN LIPIDOL., vol. 5, no. 6, 1 December 2010 (2010-12-01), pages 838 - 852 *
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